American Journal of Botany 96(2): 519–530. 2009.

F AMILIAL PLACEMENT AND RELATIONS OF REHMANNIA AND TRIAENOPHORA (SCROPHULARIACEAE S.L.) INFERRED FROM FIVE GENE REGIONS 1

Zhi Xia, 2 Yin-Zheng Wang,2,4 and James F. Smith3

2 State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, 20 Nanxincun, Xiangshan, Beijing 100093, China; and 3 Department of Biological Sciences, Boise State University, 1910 University Drive, Boise, Idaho 83725 USA

Accurate classifi cation systems based on evolution are imperative for biological investigations. The recent explosion of molecular phylogenetics has resulted in a much improved classifi cation of angiosperms. More than fi ve phylogenetic lineages have been recog- nized from Scrophulariaceae sensu lato since the family was determined to be polyphyletic; however, questions remain about the genera that have not been assigned to one of the segregate families of Scrophulariaceae s.l. Rehmannia Liboschitz and Triaenophora Solereder are such genera with uncertain familial placement. There also is debate whether Triaenophora should be segregated from Rehmannia . To evaluate the phylogenetic relations between Rehmannia and Triaenophora , to fi nd their closest relatives, and to verify their familial placement, we conducted phylogenetic analyses of the sequences of one nuclear DNA (ITS) region and four chloroplast DNA gene regions (trnL-F , rps16 , rbcL , and rps2 ) individually and combined. The analyses showed that Rehmannia and Triaenophora are each strongly supported as monophyletic and together are sister to . This relation was corrobo- rated by phytochemical and morphological data. Based on these data, we suggest that Rehmannia and Triaenophora represent the second nonparasitic branch sister to the remainder of Orobanchaceae (including Lindenbergia ).

Key words: DNA sequence; familial placement; Orobanchaceae; phylogenetic relations; Rehmannia ; Scrophulariaceae sensu lato; Triaenophora .

Nothing in biology makes sense except in the light of corolla shape and fruit dehiscence ( Fischer and Meyer, 1835 ). evolution. — Theodosius Dobzhansky (1964 ) Since then, Rehmannia has usually been placed in the tribe Digitaleae in Scrophulariaceae s.l. ( Bentham, 1876 ; Solereder, As a corollary to Theodosius Dobzhansky’ s famous quote, 1909 ; Li, 1948 ; Chin, 1979 ). Others have suggested Rehmannia understanding the evolutionary history of organisms can improve to be part of subfamily Cyrtandroideae in based our understanding of biology. Recent molecular phylogenetic on reports of its unilocular ovary (de Candolle, 1845; Hemsley, analyses have resulted in a major rearrangement of angiosperm 1895 ; Solereder, 1909 ; Li, 1948 ; Burtt, 1954 ). classifi cation that now better refl ects the evolutionary history Some species initially described as Rehmannia have been of these . However, many species remain unsampled segregated as monotypic, i.e., Triaenophora and Titanotrichum and thus unplaced in the new classifi cation scheme. A series of (Solereder, 1909). Titanotrichum was transferred to Gesneriaceae molecular systematic studies of Scrophulariaceae s.l. have ( Solereder, 1909 ), a move that is supported by both morphologi- revealed that the traditionally circumscribed Scrophulariaceae cal and molecular data (Burtt, 1954; Wang et al., 1990, 1992, 2002, is polyphyletic ( Olmstead and Reeves, 1995 ; dePamphilis et al., 2004 ; Smith et al., 1997a , b ; Pan et al., 2002 ). On the contrary, 1997 ; Olmstead et al., 2001 ; Oxelman et al., 2005). These studies Triaenophora , which now contains three species ( Solereder, have resulted in recircumscriptions and new descriptions of 1909; Chin, 1979; Li et al., 2005), has received almost no atten- families to encompass the monophyletic lineages that were tion besides Li (1948) who returned it to Rehmannia , and Chin recovered. However, questions remain about the genera that (1979) who later segregated it. have not been assigned to one of the segregate families of As for the phylogenetic relations of Rehmannia and Triaeno- Scrophulariaceae s.l., such as Rehmannia and Triaenophora . phora, one issue is whether there is any phylogenetic affi nity The genus Rehmannia Liboschitz consists of six species with Digitalis ( Gaertner, 1770 ). Digitalis , and Rehmannia / endemic to China (Chin, 1979), in which R. glutinosa is widely Triaenophora are remarkably different from each other in their distributed in central China and cultivated in Japan and Korea corolla shapes and a series of morphological characters such ( Rix, 1987 ) and is an important species in traditional Chinese as infl orescence morphology and fruit dehiscence, as well as medicine. Rehmannia has longstanding controversies surround- geographic distribution ( Chin, 1979 ; Wang and Wang, 2005 ). ing its systematic placement at both the generic and familial All phylogenetic analyses of Scrophulariaceae s.l. have placed levels. It was originally included within Digitalis ( Gaertner, Digitalis in Plantaginaceae (Olmstead et al., 2001; APG II, 1770) and was established by Liboschitz in 1835 because of its 2003; Albach et al., 2005; Oxelman et al., 2005; Tank et al., 2006). A second, equally likely probability is that Rehmannia and Triaenophora are members of Gesneriaceae because Titan- 1 Manuscript received 13 June 2008; revision accepted 10 October 2008. otrichum, a segregate from Rehmannia, has been convincingly The authors thank Professor De-Yuan Hong for helpful comments on the manuscript. This study was supported by the National Natural Science placed there (Burtt, 1954; Wang et al., 1990, 1992, 2002, 2004; Foundation of China Grant 30570105, and CAS Grant KSCX2-YW-R-135. Smith et al., 1997a , b ; Pan et al., 2002 ). 4 Author for correspondence (e-mail: [email protected]) Recently, Rehmannia was included in a cladistic analysis of DNA sequence data for the further disintegration of Scrophula- doi:10.3732/ajb.0800195 riaceae, in which a single species of Rehmannia was sister to 519 520 American Journal of Botany [Vol. 96

Lancea and Mazus (subfamily Mazoideae of Phyrmaceae sensu 1997 ), respectively. PCR products were purifi ed with the UNIQ-10 PCR purifi - Beardsley and Olmstead, 2002 ) and in a clade with Paulownia , cation kit (Sangon, Shanghai, China). Sequencing primers were the same as amplifi cation primers. Automated sequencing was performed on a MegaBACE Orobanchaceae, and Phrymaceae ( Oxelman et al., 2005 ). Sam- 1000 automatic sequencer (Amersham Biosciences, Sunnyvale, California, pling all six species of Rehmannia, Albach et al. (2007), using USA) using manufacturer ’ s protocols. The DNA sequences reported in the pa- nuclear ITS and chloroplast trnL-F and rps16 sequences, placed per have been deposited in GenBank with accession numbers shown in Table 1. Lindenbergia as sister to Rehmannia . However, the low sampling For some genera, sequences of the fi ve regions sampled here were available of species other than Rehmannia weakens their result (only four only for different species. Rather than limit our sampling of either genera or species including outgroups are sampled outside Rehmannia ). sequences, we combined sequences from different species into a single genus in Rehmannia our analyses provided that there was evidence that the genus was monophyletic As we have outlined, the familial placement of and the genus was not the primary focus of our study. and Triaenophora has not been well resolved either from a morphological or a molecular perspective. The debate regard- Sequence alignment and phylogenetic analysis— Sequence alignments ing the familial placement of Rehmannia and Triaenophora in were made with the program CLUSTAL_X (Thompson et al., 1997) and refi ned morphology is mainly based on the selection of characters used manually for the maximization of sequence homology using the program for classifi cation ( Solereder, 1909 ; Burtt, 1954 ), many of which BioEdit 5.0.9 ( Hall, 1999 ). have been revealed to be convergent (Olmstead et al., 2001) Parsimony analysis for each matrix was carried out using maximum parsi- and thus provide little insight regarding the evolutionary rela- mony (MP) methods in the program PAUP* version 4.0b10 ( Swofford, 2002 ). All characters and character-state changes were specifi ed as unordered and tions for the classifi cation system. Meanwhile, the controversy weighted equally, and gaps were coded as missing data. Heuristic searches in molecular data lies in the lack of suffi cient sampling among were performed with 1000 replicates of random addition, one tree held at each the putative relatives of Rehmannia and Triaenophora and the step during stepwise addition, tree-bisection-reconnection (TBR) branch swap- relation between these two genera. ping, Multrees in effect, and steepest descent off. To examine the robustness Greater sampling of taxa putatively close to Rehmannia and of various clades, we ran a bootstrap analysis (Felsenstein, 1985) with 500 Triaenophora and the analysis of additional DNA regions are replicates using a heuristic search with 1000 replicates of random sequence addition and TBR branch swapping. necessary to determine the familial placement of these two gen- Bayesian inference (BI) was conducted using the program MrBayes version era and their closest relatives in s.l. This study is thus 3.0b4 (Ronquist and Huelsenbeck, 2003). The program Modeltest 3.06 (Posada conducted with a comprehensive sampling of putative relatives and Crandall, 1998 ) was employed to determine the appropriate model of se- of Rehmannia and Triaenophora in Lamiales s.l. and the use of quence evolution for each DNA data set. Four chains of Markov chain Monte fi ve DNA regions (ITS, trnL-F , rps16 , rbcL, and rps2) that have Carlo (MCMC) were each run for 10 000 000 generations and were sampled been shown to be particularly informative in the Lamiales s.l. every 100 000 generations, starting with a random tree. For each run, the fi rst 50 samples before the chains reached stationarity were discarded as burn-in. (dePamphilis et al., 1997; Smith et al., 1997a, b ; Nickrent et al., Posterior probability (PP) was used to estimate robustness. 1998; Young et al., 1999; Olmstead et al., 2001; Beardsley and For combined sequence data, the incongruence-length-difference (ILD) test Olmstead, 2002; Albach et al., 2005; Oxelman et al., 2005; ( Farris et al., 1994 ) was conducted, using the partition homogeneity test in Wolfe et al., 2005 ; Tank et al., 2006 ). The goal of this study was PAUP* 4.0b10 (Swofford, 2002), to examine the congruence between nuclear to (1) evaluate the phylogenetic relation between Rehmannia ITS and chloroplast data sets. Test settings were 100 random stepwise additions and Triaenophora , (2) fi nd their closest relatives, and thereby and 1000 replicates of heuristic search with TBR branch swapping ( Farris et al., 1994 ). The resulting P value was used to determine whether the two data sets (3) verify their familial placement. had signifi cant incongruence (P < 0.05). We excluded species from the com- bined data set if they only had ITS or cpDNA sequences available. Topological congruence between the trees constraining Phrymaceae as MATERIALS AND METHODS monophyletic and no constraint was evaluated with the Templeton (1983) test using PAUP* 4.0b10 ( Swofford, 2002 ). The phylogenetic analysis herein was Taxon sampling —We sampled all six species of Rehmannia and two divided into two steps as follows. We fi rst conducted cladistic analyses of com- of three species of Triaenophora . To fully examine the putative relatives of bined cpDNA ( trnL-F , rps16 , rbcL, and rps2) from all sampled taxa. The four Rehmannia and Triaenophora, we selected one species of Paulownia, two gen- chloroplast regions (cpDNA) included in this study formed a single linkage era of Gesneriaceae, six genera of Plantaginaceae (including Digitalis ), seven group as part of the chloroplast genome, so confl icts that might arise between genera of Scrophulariaceae sensu stricto, six genera of Phyrmaceae (four in data partitions from different sources subject to different evolutionary histories Phrymoideae and two in Mazoideae including four species of Mazus in addition should not exist ( Olmstead et al., 2001 ). The genus Calceolaria was selected to the one species sampled in previous studies), 12 representative genera of four as the outgroup based on Olmstead et al. (2001) . Attempts to use ITS sequences major clades in Orobanchaceae sensu lato (including the nonparasitic genus at this level resulted in numerous ambiguities, and analyses of these sequences Lindenbergia ), three genera of Acanthaceae, two genera of Bignoniaceae, one resulted in spurious relations among some taxa. Second, based on the described genus of Lamiaceae, and one genus of Pedaliaceae. Taxon sampling was based analyses, together with results of previous molecular systematic studies on recent molecular systematic studies (dePamphilis et al., 1997; Smith et al., (Beardsley and Olmstead, 2002; Oxelman et al., 2005), we selected different 1997a, b ; Nickrent et al., 1998; Young et al., 1999; Olmstead et al., 2001; taxa for separate and combined analyses of nrDNA ITS and combined cpDNA Beardsley and Olmstead, 2002 ; Albach et al., 2005 ; Oxelman et al., 2005 ; (e.g., excluding Scrophulariaceae s.s., Plantaginaceae, Gesneriaceae), focusing Wolfe et al., 2005 ; Bennett and Mathews, 2006 ; Tank et al., 2006 ). Voucher on clades presumably closest to Rehmannia/Triaenophora and Orobanchaceae. specimens are deposited in the Herbarium of the Institute of Botany, Chinese The representative species of Acanthaceae, Bignoniaceae, Lamiaceae, and Academy of Sciences (PE). The materials studied and details of voucher speci- Pedaliaceae were selected as the outgroups. mens are shown in Table 1 .

DNA extraction, PCR amplifi cation, and sequencing— Total DNA was RESULTS extracted from silica-gel-dried material using the CTAB method following the protocol of Rogers and Bendich (1988) and used as the template in the A comparison of the sequences we generated with those pub- polymerase chain reaction (PCR). The entire ITS region, comprising ITS1, 5.8S lished from Albach et al. (2007) indicates that the ITS sequences rDNA, and ITS2, was amplifi ed with primers ITS1 and ITS4 (Wendel et al., are identical to each other, the rps16 sequences were 99.76– 1995 ). The trnL-F region was amplifi ed with primers c and f of Taberlet et al. 100% similar, and the trnL - F sequences were 99.29 – 100% (1991) . The rps16 intron was amplifi ed with primers rps16 -2F and rps16- R3 (Bremer et al., 2002). The rbcL and rps2 gene regions were amplifi ed with similar. These comparisons confi rm the accuracy of both the primers RH1 and Z1352R (Olmstead and Reeves, 1995; Wolfe and dePamphi- sequences generated herein and those of Albach et al. (2007) lis, 1997 ; Olmstead et al., 2001 ), and rps2 -18F and rps2 -661R (dePamphilis et al., for Rehmannia . February 2009] Xia et al. — Familial placement of R EHMANNIA and TRIAENOPHORA 521

Table 1. Species, voucher with collection locality and GeneBank accession number for taxa included in this study (new sequences are in boldface). NA: not applicable; PE: Herbarium, Institute of Botany, Chinese Academy of Sciences where the voucher specimens were deposited.

GenBank accession number Taxon Voucher, collection locality and citation ITS trnL-F rps16 rbcL rps2

Ingroups Rehmannia R. chingii H. L. Li XZ-2004 – 04 – 004; Zhejiang, China. (PE)EF363673 EF363679 FJ172696 FJ172724 FJ172710 R. elata N. E. Brown Albach et al., 2007 DQ069315 DQ856496 DQ856490 — — R. glutinosa (Gaert.) Libosch. XZ-2004 – 04 – 005; Beijing, China. (PE)EF363674 EF363680 FJ172697 FJ172725 FJ172711 ex Fisch. et Mey. R. henryi N. E. Brown XZ-2004 – 04 – 002; Hubei, China. (PE)EF363671 EF363677 FJ172694 FJ172722 FJ172708 R. piasezkii Maximowicz XZ-2004 – 04 – 001; Hubei, China. (PE)EF363670 EF363676 FJ172693 FJ172721 FJ172707 Rehmannia solanifolia XZ-2004 – 04 – 003; Chongqing, EF363672 EF363678 FJ172695 FJ172723 FJ172709 Tsoong et Chin China. (PE) Triaenophora T. rupestris (Hemsl.) Solereder XZ-2004 – 04 – 009; Hubei, China. (PE)EF363675 EF363681 FJ172698 FJ172726 FJ172712 T. shennongjiaensis X. D. Li XZ-2007 – 0522; Hubei, China. (PE)FJ172741 FJ172690 FJ172704 FJ172732 FJ172717 Acanthaceae Barleria lupulina Lindl. McDade et al., 2000 AF169751 — — — — B. prionitis Lindl. Olmstead et al., 2001 — — — L01886 AF248247 Elytraria crenata Vahl. Olmstead et al., 2001 — — — AF188127 — E. imbricata Vahl. McDade et al., 2000 AF169852 AF061819 — — — Thunbergia alata Bojer McDade et al., 2000; Olmstead AF169850 AJ608564 AJ609131 — AF248248 ex Sims et al., 2001; Oxelman et al., 2005 T. usumbarica Lindau Olmstead et al., 2001 — — — L12596 — Bignoniaceae Catalpa speciosa Warder Oxelman et al., 2005; Li, 2008 AY486307 AJ608599 AJ609197 — — ex Engelm Catalpa sp. Olmstead et al., 2001 — — — L11679 AF248256 Kigelia africana Benth. Olmstead et al., 2001; Gutierrez AY178638 — — AF102648 U48764 and Freeman, unpublished data Gesneriaceae Streptocarpus caulescens Vatke Oxelman et al., 2005 NA AJ608601 AJ609135 — — Streptocarpus holstii Engl. Olmstead et al., 2001 NA — — L14409 — Titanotrichum oldhamii Wang et al., 2004 NA AY423129 — AF206829 — (Hemsl) Solereder. Lamiaceae Lamium purpureum L. Olmstead et al., 2001; Oxelman AB266244 AJ608588 AJ609175 U75702 AF248259 et al., 2005; Sudarmono and Okada, unpublished data Orobanchaceae Alectra sessilifl ora Benth. Olmstead et al., 2001; Wolfe et al., 2005 AY911210 — — AF026820 U48742 Boschniakia strobilacea Olmstead et al., 2001; Wolfe et al., 2005 AY911215 — — AF26818 U48758 A. Gray Buchnera glabrata Benth Wolfe et al., 2005 AY911216 NA NA NA NA Castilleja linariifolia Benth Olmstead et al., 2001; Tank — — EF103788 AF026823 U48739 and Olmstead, 2008 Castilleja sulphurea Rydberg Beardsley and Olmstead, 2002 AF478944 AF479008 — — — Lindenbergia philippensis Olmstead et al., 2001; Oxelman AY911231 AJ608586 AJ609169 AF123664 AF055151 Benth. et al., 2005; Wolfe et al., 2005 Melampyrum lineare Lam. Olmstead et al., 2001; Jobson and — AF482608 — AF026834 — Albert, 2002 M. sylvaticum L. Olmstead et al., 2001; Wolfe et al., 2005 AY911232 — — — AF055148 Melasma scabrum Berg. Olmstead et al., 2001; Wolfe et al., 2005 AY911233 — — AF190904 U48743 corymbosa Olmstead et al., 2001; Wolfe et al., 2005 AY911236 — — U73969 U48760 (Rydb.) Ferris Orobanche hederae Duby Bremer et al., 2002 — — AJ431050 — — O. minor Sm. Lohan and Wolfe, 1998 — AJ007724 — — — Pedicularis attollens (A.) Gray Tank and Olmstead, 2008 — EF103899 EF103821 — — P. foliosa L. Olmstead et al., 2001; Ree, 2005 AY949679 — — AF026836 U48740 Schwalbea americana L. Wolfe et al., 2005 AY911252 NA NA NA NA Seymeria laciniata Standl. Tank and Olmstead, 2008 — EF103898 EF103820. — — S. pectinata Pursh Olmstead et al., 2001; Wolfe et al., 2005 AY911253 — — AF026837 AF055141 Tozzia alpina L. Olmstead et al., 2001; Wolfe et al., 2005 AY911258 — — AF026843 U48754 Paulownia P. tomentosa (Thunb.) Steud Beardsley and Olmstead, 2002; AF478941 AF479005 AJ609153 L36447 AF055155 Oxelman et al., 2005 Pedaliaceae Sesamum indicum L. Olmstead et al, 2001; Beardsley and AF478946 AF479010 AJ609226 L14408 AF248261 Olmstead, 2002; Oxelman et al., 2005 522 American Journal of Botany [Vol. 96

Table 1. Continued.

GenBank accession number Taxon Voucher, collection locality and citation ITS trnL-F rps16 rbcL rps2 Phrymaceae Berendtia laevigata Oxelman et al., 2005 — AJ608615 AJ609208 — — B. L. Rob et Greenm. B. rugosa (Benth.) Gray Beardsley et al., 2004 AY575398 — — — — Hemichaena fruticosa Benth. Beardsley and Olmstead, 2002; AF478921 AJ608632 AJ609179 — — Oxelman et al., 2005 Lancea tibetica XZ-2007 – 0525; Sichuan, China. (PE)FJ172736 FJ172685 FJ172699 FJ172727 FJ172713 Hook. f. et Thoms. Mazus gracilis Hemsl. XZ-2007 – 058; Henan, China. (PE)FJ172738 FJ172687 FJ172701 FJ172729 FJ172715 M. japonicus XZ-2007 – 051; Beijing, China. (PE)FJ172737 FJ172686 FJ172700 FJ172728 FJ172714 (Thunb.) O. Kuntze. M. omeiensis Li. XZ-2007 – 0515; Sichuan, China. (PE)FJ172739 FJ172688 FJ172702 FJ172731 — M. reptans N. E. Br. Beardsley and Olmstead, 2002 AF478940 NA NA NA NA M. spicatus Vaniot. XZ-2007 – 0514; Henan, China. (PE)FJ172740 FJ172689 FJ172703 FJ172730 FJ172716 M. stachydifolius Oxelman et al., 2005 — AJ607432 AJ609167 — — (Turcz.) Maxim AJ607433 Mimulus aurantiacus Curtis Olmstead et al., 2001; Beardsley and AF478917 AF478982 AJ609163 AF026835 AF055154 Olmstead, 2002; Oxelman et al., 2005 M. tenellus var . tenellus Bunge XZ-2007 – 053; Henan, China. (PE)FJ172742 FJ172691 FJ172705 FJ172733 FJ172718 Mimulus szechuanensis Pai XZ-2007 – 0523; Sichuan, China. (PE)FJ172743 FJ172692 FJ172706 FJ172734 FJ172719 Phryma leptostachya L. Oxelman et al., 2005 — AJ430928 AJ609150 — — P. leptostachya Beardsley and Olmstead, 2002; AF478924 — — FJ172735 FJ172720 L. var asiatica Hara XZ-2007 – 061; Henan, China. (PE) Plantaginaceae Antirrhinum majus L. Olmstead et al., 2001; NA AJ608634 AJ609218 L11688 U48766 Oxelman et al., 2005 Chelone obliqua L. Olmstead et al., 2001; Oxelman NA DQ531203 AJ609220 AF026824 U48770 et al., 2005; Wolfe et al., 2006 Collinsia grandifl ora Lindley Olmstead et al., 2001 NA — — AF026825 AF248252 C. heterophylla R.Grah Wolfe et al., 2006 NA DQ531198 — — — C. tinctoria Hartw. ex. Benth Albach et al., 2005 NA — AY492200 — — Digitalis obscura L. Albach et al., 2004; Albach NA AF486418 AY218799 — — and Chase, 2004 D.s purpurea L. Olmstead et al., 2001 NA — — L01902 U48767 Plantago coronopus L. Albach and Chase, 2004 NA — AY218801 — — P. lanceolata L. Olmstead et al., 2001; NA AY101952 — L36454 — R ø nsted et al., 2002 P. major L. Olmstead et al., 2001 NA — — — AF248254 Veronica arvensis L. Olmstead et al., 2001 NA — — — U48768 V. persica Poir. Olmstead et al., 2001; NA AF513336 — L36453 — Albach et al., 2004 V. campylopoda Boiss. Albach and Chase, 2004 NA — AY218811 — — Scrophulariaceae s.s. Alonsoa unilabiata Olmstead et al., 2001; NA AJ608620 AJ609217 AF026821 AF248262 (L. f.) Steud. Oxelman et al., 2005 Buddleja davidii Franchet Olmstead et al., 2001; NA AJ608612 AJ609204 L14392 AF248264 Oxelman et al., 2005 Leucophyllum frutescens Olmstead et al., 2001; NA AF380873 AJ609171 AF123665 AF055156 I. M. Johnston Oxelman et al., 2005 Myoporum mauritianum Olmstead et al., 2001; NA AJ608582 AJ609161 L36445 — A. DC. Oxelman et al., 2005 M. parvifolium R.Br. Olmstead et al., 2001 NA — — — AF055157 Nemesia strumosa Benth. Olmstead et al., 2001; NA AJ608631 AJ609159 AF123663 AF248265 Oxelman et al., 2005 Scrophularia californica Olmstead et al., 2001; NA — AJ609224 L36449 U48762 Cham. & Schldl. Oxelman et al., 2005 Scrophularia ningpoensis Chen et al., 2005 NA AY695886 — — — Hemsl. Verbascum arcturus L. Oxelman et al., 2005 NA — AJ609128 — — V. blattaria L. Olmstead et al., 2001 NA — — — U48763 V. thapsus L. Olmstead et al., 2001 NA — — L36452 — V. speciosum Schrad. Mayer et al., 2003 NA AJ492271 — — — Outgroup Calceolariaceae Calceolaria mexicana Benth Olmstead et al., 2001; NA AJ608611 AJ609202 AF123669 AF055162 Oxelman et al., 2005 February 2009] Xia et al. — Familial placement of R EHMANNIA and TRIAENOPHORA 523

Analyses with all sampled taxa— Data for one or two of the [BS] = 94%; posterior probability [PP] = 100%). Clades B and four chloroplast regions are missing for 15 genera, but no genus C contained the species of Plantaginaceae (BS = 91%; PP = was missing more than two sequences of the four genes. 100%) and Scrophulariaceae sensu stricto (BS = 87%; PP = The entire cpDNA data set consists of 4125 bp, of which 2554 100%), respectively. Clade D was Acanthaceae, Pedaliaceae, (61.9%) were constant, 721 (17.5%) were variable but uninfor- Lamiaceae, and Bignoniaceae (BS = 73%, PP = 100%) and mative, and 850 (20.6%) were parsimony informative. Parsi- clade E was Mazoideae (BS = PP = 100%). Clade F comprised mony analyses resulted in nine trees of 3511 steps each Phrymoideae with BS = PP = 100%. Clade G included (consistency index [CI] = 0.623; retention index [RI] = 0.608). Rehmannia , Triaenophora, and Orobanchaceae with BS = 62% One most parsimonious (MP) tree of cpDNA data (Fig. 1) was and PP = 99%. Rehmannia and Triaenophora formed one congruent with the Bayesian tree (the best-fi t model GTR + I + strongly supported lineage (BS = 100%; PP = 100%) and was G) in topology. The MP tree comprised six main clades labeled sister to Orobanchaceae, that was likewise strongly supported A – G. Titanotrichum oldhamii was sister to Streptocarpus in as monophyletic (BS = 88%; PP = 100%). Paulownia was sister Gesneriaceae (clade A) with high support (bootstrap support to clade G with low support.

Fig. 1. One of nine most parsimonious trees generated from analysis of combined chloroplast for all sampled taxa. Branch lengths are proportional to number of nucleotide substitutions (scales represent 10 substitutions). Bootstrap (BS) values (≥ 50%) are above the branches; Bayesian posterior probabilities (PP) ( ≥ 90%) are below the branches. Maz = Mazus , Mim = Mimulus , Rehm = Rehmannia , Triaen = Triaenophora . 524 American Journal of Botany [Vol. 96

Analyses with selected taxa— ITS analysis — The aligned se- tree (the best-fi t model GTR + I + G) in topology except for the quences of ITS had 656 bp, of which 209 (31.9%) were con- position of Paulownia . Paulownia was sister to Phrymoideae stant, 94 (14.3%) were variable but uninformative, and 353 (clade F) in the MP tree, but sister to the group that includes (53.8%) were parsimony informative. Parsimony analysis re- clades E, F, and G in the Bayesian tree. The ITS MP tree com- sulted in 10 trees of 2004 steps each, CI of 0.445, and RI of prised three main clades labeled as E, F, and G, that correspond 0.591. The MP tree ( Fig. 2 ) was congruent with the Bayesian to the clades recovered in Fig. 1 . Rehmannia , Triaenophora ,

Fig. 2. One of 10 most parsimonious trees generated from the ITS data for selected taxa. Branch lengths are proportional to number of nucleotide substitutions (scales represent 10 substitutions). Branches marked by an asterisk indicate the topological discordance between most parsimonious (MP) and Bayesian trees. Bootstrap (BS) values (≥ 50%) are above the branches; Bayesian posterior probabilities (PP) ( ≥ 90%) are below the branches. Maz = Mazus , Mim = Mimulus , Rehm = Rehmannia , Triaen = Triaenophora . February 2009] Xia et al. — Familial placement of R EHMANNIA and TRIAENOPHORA 525

Paulownia, Mazoideae, Phrymoideae, and Orobanchaceae DISCUSSION formed a monophyletic group with maximum support. Clade E was Mazoideae, and clade F comprised all sampled species of Phylogenetic relation between Rehmannia and Triaenophora— Phrymoideae with BS = 60% and PP = 100%. Clade G was also Triaenophora has been considered closely related to Rehmannia recovered as monophyletic including Rehmannia , Triaeno- in traditional systematics (Forbes and Hemsley, 1890; Solereder, phora, and Orobanchaceae with BS = 92% and PP = 100%. 1909; Li, 1948; Chin, 1979). Our molecular data show that Rehmannia and Triaenophora, each as a monophyletic group, Rehmannia and Triaenophora form a strongly supported clade, formed one strongly supported lineage (BS = 89%; PP = 94%), in which Triaenophora is sister to Rehmannia . The sister rela- which was sister to Orobanchaceae. Orobanchaceae was like- tion between Rehmannia and Triaenophora is also corroborated wise strongly supported as monophyletic (BS = 80%; PP = by allozymic variability ( Li et al., 2007 ) and numerical analysis 100%). The low supports for the relations within Orobanchaceae of morphological data (Li et al., 2008). This sister relation is not were probably due to the sparse taxon sampling within the surprising because Rehmannia and Triaenophora share a series family. Templeton ’ s test indicated incongruence between the of uniform synapomorphies, such as two lateral bracteoles at trees constraining Phrymaceae (Phrymoideae and Mazoideae) the base of the pedicel just above the subtending bract (they are as monophyletic and that no constraint was insignifi cant aborted early in development in R. chingii , R. solanifolia , and ( P = 0.6103). R. glutinosa), four stamens with a gap at the expected site of the adaxial staminode, and the unidirectional initiation of corolla Analysis of combined chloroplast data — The combined chlo- lobes and stamens from the abaxial to the adaxial side ( Wang roplast data set consisted of 3880 positions, of which 2783 and Wang, 2005). Triaenophora has a series of unique traits (71.7%) were constant, 556 (14.4%) were variable but uninfor- distinctive from those of Rehmannia , i.e., fi ve trifi d calyx lobes; mative, and 541 (13.9%) were parsimony informative. Parsi- dense, white, spreading, lanose-villous hairs on the stems, mony analyses resulted in six trees of 1845 steps each (CI = , and pedicels; and a bilocular ovary ( Chin, 1979 ; Wang 0.73; RI = 0.737). The MP tree of cpDNA data (Fig. 3) was and Wang, 2005 ). Rehmannia, as a strongly supported mono- congruent with the Bayesian tree (the best-fi t model GTR + I + phyletic group distinct from Triaenophora, is characterized G) in topology. Clades E, F, and G, found in the ITS tree ( Fig. 2 ), by fi ve revolute and undivided calyx lobes; brown or white were also recovered in the cpDNA tree with high support glandular hairs on stems, leaves, and pedicels; and one ovarian (Fig. 3). The topology of the cpDNA MP tree (Fig. 3) differed locule ( Chin, 1979 ; Wang and Wang, 2005 ). from the ITS topology ( Fig. 2 ) mainly in the position of Pau- Our results regarding the relations among species within lownia . Paulownia was sister to Phrymoideae (Clade F) with Rehmannia are in agreement with Albach et al. (2007) and Li low support in the ITS tree (Fig. 2), but was sister to clade G et al. (2007 , 2008 ). Albach (D. C. Albach, Johannes Gutenberg- with low support in the cpDNA tree ( Fig. 3 ). Clade G, which Universitä t Mainz, Germany, unpublished data) conducted a includes Rehmannia , Triaenophora , and Orobanchaceae, was study similar to ours with the exception that a single species of recovered as monophyletic with BS = 59% and PP = 100%. Triaenophora was included (T. rupestris ) and in place of rps2 , Rehmannia and Triaenophora, each as a monophyletic group, analyzed ndhF sequences. The results of these two indepen- formed one maximum supported lineage, which was sister to dently conducted studies provide mutual confi rmation that Orobanchaceae. Orobanchaceae was strongly supported as Triaenophora and Rehmannia are sister to each other and to- monophyletic (BS = 78%; PP = 100%) in which Lindenbergia gether are sister to Orobanchaceae. Likewise, both studies fi nd was sister to the remainder of Orobanchaceae. Clade G and evidence against the monophyly of Phyrmaceae (discussed Paulownia were further clustered with Phrymoideae (clade F) later). Our results further show that the monotypic genus Titan- with moderate support. Templeton’ s test indicated incongru- otrichum initially described as a species of Rehmannia, is not ence between the trees constraining Phrymaceae as monophyl- closely related, but better included in Gesneriaceae (Burtt, etic and that no constraint was insignifi cant (P = 0.5316). 1954 ; Wang et al., 1990 , 1992, 2002, 2004 ; Smith et al., 1997a , b ; Pan et al., 2002 ). Analysis of combined chloroplast and nuclear ITS data— The ILD test gave a value of P = 0.21, indicating that the data Familial placement of Rehmannia and Triaenophora— sets were not signifi cantly different from random partitions of Rehmannia and Triaenophora were traditionally placed in the the combined chloroplast and ITS data. The combined chloro- Digitaleae (Scrophulariaceae s.l.) with close affi nity to Digitalis plast and ITS data sets consisted of 4536 positions, of which ( Bentham, 1846 , 1876 ; Forbes and Hemsley, 1890 ; Wettstein, 2998 (66.1%) were constant, 653 (14.4%) were variable but 1891 ; Li, 1948 ; Chin, 1979 ). However, the inclusion of Reh- uninformative, and 885 (19.5%) were parsimony informative. mannia within Digitaleae was questioned when Oxelman et al. Parsimony analyses resulted in one tree of 3735 steps (CI = (2005) placed one species of Rehmannia as sister to Mazus and 0.592, RI = 0.652). The MP tree (Fig. 4) was congruent with the Lancea (Scrophulariaceae s.l. or Mazoideae sensu Beardsley and Bayesian tree (the best-fi t model GTR + I + G) in topology. The Olmstead, 2002 ). In all trees herein, Rehmannia and Triaenophora topology of the MP tree (Fig. 4) from combined cpDNA and are shown to not have a close affi nity with any Digitaleae. Our nuclear ITS data were completely congruent with the ITS MP results also show no close relation between Rehmannia and tree in the major clades ( Fig. 2 ) and the cpDNA MP tree except Gesneriaceae including Titanotrichum, a relation that has been for the position of Paulownia in the cpDNA tree (Fig. 3). The traditionally suggested (de Candolle, 1845; Hemsley, 1895; ingroup nodes in the topology of the combined chloroplast and Solereder, 1909 ; Li, 1948 ; Burtt, 1954 ). ITS data received higher support than those in the separate Rehmannia and Triaenophora , as a monophyletic group, are analyses of either cpDNA or nuclear ITS data alone. Temple- shown herein to be sister to Orobanchaceae (including Linden- ton ’ s test indicated incongruence between the trees constrain- bergia ) with moderate to high support BS = 92 for ITS, 59 – 62 ing Phrymaceae as monophyletic and that no constraint was for cpDNA, 94 for combined and high to maximum PP (99 –100) insignifi cant (P = 0.4054). from all analyses. Lindenbergia is sister to other parasitic genera 526 American Journal of Botany [Vol. 96

Fig. 3. One of six most parsimonious trees generated from combined chloroplast data for selected taxa. Branch lengths are proportional to number of nucleotide substitutions (scales represent 10 substitutions). Bootstrap (BS) values ( ≥50%) are shown above the branches, and Bayesian posterior probabili- ties (PP) values (≥ 90%) are indicated below the branches. Maz = Mazus , Mim = Mimulus , Rehm = Rehmannia , Triaen = Triaenophora . of Orobanchaceae with high support in trees of cpDNA and sitic genus Lindenbergia ( dePamphilis et al., 1997 ; Nickrent combined ITS and cpDNA data as seen in previous molecular et al., 1998 ; Wolfe and dePamphilis, 1998 ; Young et al., 1999 ; phylogenies ( Young et al., 1999 ; Olmstead et al., 2001 ; Wolfe Olmstead et al., 2001 ; Wolfe et al., 2005 ; Bennett and Mathews, et al., 2005; Bennett and Mathews, 2006; Tank et al., 2006) 2006; Tank et al., 2006). The sister relationship between Reh- rather than sister to Rehmannia and Triaenophora ( Albach mannia and Mazoideae recognized in Oxelman et al. (2005) et al., 2007). Our results further confi rm that Orobanchaceae is is not supported by our molecular data with greater sampling a well-supported lineage that includes the holoparasitic members both in Rehmannia and Mazoideae. Rehmannia and Triaeno- traditionally treated in Orobanchaceae, the hemiparasitic taxa phora together with Orobanchaceae are sister to Paulownia and previously treated under Scrophulariaceae s.l. and the nonpara- Phrymoideae. February 2009] Xia et al. — Familial placement of R EHMANNIA and TRIAENOPHORA 527

Fig. 4. Single most parsimonious tree generated from combined chloroplast and ITS data for selected taxa. Bootstrap (BS) values (≥ 50%) are above the branches; Bayesian posterior probabilities (PP) (≥ 90%) are below the branches. Maz = Mazus , Mim = Mimulus , Rehm = Rehmannia , Triaen = Triaeno- phora . ᭿ part of capsule exserted from the persistent calyx tube; ٗ capsule included in persistent calyx tube; ‡ capsule almost completely exserted from the persistent calyx tube; ᭹ seeds with alveolate and pitted testa; { seeds with smooth or anomalous reticulate surfaces; | seeds with membranous wings; ᭡ calyx of connate sepals with spinescent apices; ᭝ calyx with long triangular and lanceolate calyx lobes; ᭜ corolla with two highly reduced and triangular upper lobes; ᭛ corolla with two oblong and orbicular upper lobes.

On the basis of observations of morphological characters, coat characters are seldom used for systematic analyses at together with previous reported data ( Chin, 1979 ; Zhang, 1990a ), higher taxonomic levels. However, seed coat characters may we conclude that Rehmannia , Triaenophora, and Orobanchaceae provide synapomorphies for some of the relations recovered in often have capsules that are half or partly exserted from the our molecular based trees. Rehmannia and Triaenophora are persistent calyx tubes, whereas in Phrymaceae, the capsules are characterized by numerous minute seeds with alveolate and re- completely included in the persistent calyx tube ( Fig. 4 ). Seed ticulate testa similar to seeds of Orobanchaceae with alveolate, 528 American Journal of Botany [Vol. 96 pitted and reticulate testa (except for seeds of Melampyrum In comparison to other groups in Lamiales s.l. with respect to with smooth testa; Fig. 4) (Musselman and Mann, 1976; Zhang, the distribution of related phytochemical and morphological char- 1990b; An and Hong, 2003; Plaza et al., 2004). Meanwhile, acters, the combination of the aforementioned features are sy- Phrymaceae possess minute seeds with smooth testa, and Pau- napomorphies for Rehmannia / Triaenophora and Orobanchaceae. lownia is characterized by seeds with membranous wings Based on the molecular results, corroborated by phytochemical ( Tsoong, 1979 ; Yang, 1979 ; Fig. 4 ). Along with Lindenbergia , and morphological data, we suggest that Rehmannia and Tri- Rehmannia, and Triaenophora have tricolporate pollen with aenophora represent the second nonparasitic branch sister to reticulate sculpting of the exine (Ying et al., 1993; Hjertson, the remainder of Orobanchaceae s.l. (including Lindenbergia ) 1995 ; Wang et al., 1997 ), which differs from the characteristic or a clade at the rank of family sister to Orobanchaceae. Our tricolpate pollen (and its variants) with retipilate sculpting of results recognizing this sister relationship represent the fi rst the exine in other parasitic genera in Orobanchaceae ( Tsoong step toward better understanding the relations of Rehmannia and Chang, 1965; Minkin and Eshbaugh, 1989; Bolliger and and Triaenophora with other segregate families of Scrophulari- Wick, 1990 ; Abu Sbaih et al., 1994 ; Zhang, 1990b ; Lu et al., aceae s.l. Further detailed studies are needed to better under- 2007 ). The evolutionary trends of pollen exine ornamentation stand morphological and anatomical synapomorphies among from reticulate to retipilate sculpting have been seen in Dicha- these species. petalaceae and in many different groups (Punt, 1976; Minkin The familial status of Paulownia and Mazus/ Lancea and Eshbaugh, 1989 ), as well as from tricolporate to tricolpate (Mazoideae) remain uncertain in the results presented here. pollen in some taxa ( Ferguson and Skvarla, 1981 ; Hong, 1984 ; Paulownia has been placed alternately in the Scrophulariaceae Minkin and Eshbaugh, 1989; Mart í nez-Ortega et al., 2000). s.l., Bignoniaceae, or assigned to a family of its own ( Nakai, The tricolpate pollen with retipilate sculpting of the exine in 1949; Beardsley and Olmstead, 2002). It is distinctively differ- parasitic genera of Orobanchaceae may be derived from tri- ent from Orobanchaceae, Scrophulariaceae s.s. and Phrymaceae colporate pollen with reticulate sculpting in the nonparasitic in its woody habit, capsule with a persistent woody calyx tube, genera Lindenbergia , Rehmannia , and Triaenophora . and seeds with membranous wings ( Fig. 4 ). The phylogenetic In addition, phytochemical characters are often used to analyses herein, with increased sampling of Mazus, indicate complement or improve molecular trees ( Grayer et al., 1999 ). that Mazus and Lancea (Mazoideae) may not be included in Iridoids have been found as natural constituents in most taxa of Phrymaceae as previously suggested by Oxelman et al. (2005) . Lamiales s.l. ( Jensen, 1992 ). Comparison of iridoid glycosides However, Templeton ’ s tests (see Results, Analyses with selected distributed among Rehmannia / Triaenophora , Orobanchaceae, taxa ) do not reject the inclusion of Mazoideae in Phrymaceae. Scrophulariaceae s.s and Plantaginaceae shows that catalpol The systematic position of Paulownia and Mazoideae deserves and aucubin are widely present in these taxa (Kitagawa et al., further detailed studies with greater taxon sampling among 1971; Oshio and Inouye, 1982; Grayer et al., 1999; Albach their putative relatives and new DNA regions together with et al., 2007 ). However, Rehmannia/Triaenophora and most genetic or evolutionary developmental methods to gain a com- Orobanchaceae (including Lindenbergia ) lack harpagide and prehensive understanding about their phylogenetic history. 6-rhamnopyranosyl-catalpol and their esters, which are char- acteristic for many Scrophulariaceae s.s. ( Jensen et al., 2008 ). 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